CN115629207A

CN115629207A - Method for treating pneumonia and heat stranguria clearing granules used by same

Info

Publication number: CN115629207A
Application number: CN202211353985.7A
Authority: CN
Inventors: 卢礼平; 唐靖雯; 潘梅; 梁斌; 张丽艳; 蒲翔; 柴艺汇; 李来来
Original assignee: Guizhou Warmen Pharmaceutical Co ltd
Current assignee: Guizhou Warmen Pharmaceutical Co ltd
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2023-01-20

Abstract

The invention relates to a method for treating pneumonia and heat stranguria clearing granules used by the method. In one aspect, it relates to a method for examining the efficacy of Relinqing particles in the treatment of pneumonia, e.g. by measuring serum TNF-alpha concentration, comprising the steps of: randomly dividing mice, atomizing with LPS for molding, and filling after moldingAdministration of Relinqing granules to the stomach, blood draw from the mice, and serum collection to determine at least one of the following: pulmonary index, TNF-alpha, IL-1 beta, LI-6, PGE in serum ₂ And (4) obtaining a statistical result of the treatment effect of the Relinqing granules on the pneumonia mice according to the detection data. The invention also provides the application of the Relinqing granules as a pneumonia treatment medicine. The invention shows that the Relinqing granules are a very excellent medicine for treating pneumonia.

Description

Method for treating pneumonia and Relinqing granules used in same

Technical Field

The invention belongs to the technical field of medicines, relates to a traditional Chinese medicine preparation, in particular to a traditional Chinese medicine preparation Relinqing granule prepared from a plant medicinal material polygonum capitatum, also relates to a preparation method of the traditional Chinese medicine preparation and pharmaceutical application thereof, and in particular relates to application of the Relinqing granule in preparing a medicine for treating pneumonia. The research result of the invention shows that the Relinqing granules have obvious positive treatment effect on pneumonia. The invention proves the effect of the Relinqing granules as the pneumonia therapeutic drug through the research on the protective effect of the mouse with acute pneumonia caused by LPS.

Background

Pneumonia (pneumonias) refers to inflammation in the lung, and is a frequently occurring and common disease of the respiratory system. Pneumonia can occur in people of any age group, but young and old people, and people with hypoimmunity or poor immune system belong to high-risk patients, and are easy to develop. If the condition is severe, it can be fatal. According to the investigation of the world health organization, the death rate of pneumonia accounts for 75% of the death rate of acute infection of the respiratory system. Pneumonia is a very broad concept, because inflammation in the lungs is called pneumonia, and its etiology can be biological, physical, chemical, etc.

The classification of pneumonia can be classified according to the origin of the infection: nosocomial infectious pneumonia and socially acquired pneumonia. (2) Pneumonia can be caused by different pathogenic factors, and can be classified into: infectious pneumonia (according to pathogen species: including bacterial pneumonia, common bacteria are streptococcus pneumoniae, staphylococcus, hemophilus influenzae, etc.; viral pneumonia, common viruses such as respiratory syncytial virus, influenza virus, parainfluenza virus, adenovirus, etc.; and also fungal pneumonia, mycoplasma pneumonia, chlamydia pneumonia, etc.), physicochemical pneumonia (such as radiation pneumonia, lipoid pneumonia of inhalation pneumonia), and allergic pneumonia (such as hypersensitivity pneumonia and rheumatalgia). (3) Due to the difference of pathogenic factors and body reactivity, the site, extent of involvement and nature of pathological changes of inflammation are different. Inflammation is called alveolar pneumonia (most pneumonia is alveolar) when the inflammation occurs in the alveoli, and interstitial pneumonia when the inflammation affects the interstitium. The range of pathological changes is referred to as lobular pneumonia in units of lobules, segmental pneumonia in which segments of the lung are involved, lobar pneumonia in which the whole or a plurality of lobules are involved, and bronchiolitis. (4) classifying according to disease course: the chronic pneumonia is classified into acute pneumonia, persistent pneumonia and chronic pneumonia, the duration of the persistent pneumonia generally reaches 1-3 months, and the chronic pneumonia is determined after more than 3 months. (5) The pathological nature can be classified into serous pneumonia, cellulosic pneumonia, suppurative pneumonia, hemorrhagic pneumonia, caseous pneumonia, granulomatous pneumonia or organizing pneumonia. Common symptoms of pneumonia include: cough with yellow-green sputum; fever with aversion to cold; chest pain, either severe or stinging, can be more severe with deep breathing or coughing; tachypnea; short gas; hyperthermia (body temperature at least 39.5 ℃).

The Relinqing granule is a single preparation taking Polygonum capitatum (Polygonum capitatum Buch. -ham. Ex D. Don) which is a characteristic medicine of Guizhou nationality as a raw material, and has the effects of clearing heat and purging fire, and inducing diuresis for treating stranguria; can be used for treating stranguria caused by damp-heat in lower-jiao with the symptoms of frequent micturition, urgent micturition, and odynuria; clinically, it is commonly used to treat diseases caused by damp-heat accumulation in the lower energizer and adverse qi transformation of the body, such as urinary tract infection and pyelonephritis [ bin, zhanliyan, ran 25035 ] and male. Chinese traditional medicine press, 2014; pharmacological action experimental study of Relinqing granules [ J ] journal of practical traditional Chinese medicine, 2012, 26 (03): 12-14; southern haifeng, liujie, wudan, etc. the clinical effect of Relinqing granules in combination with conventional antibiotics in the treatment of gonorrhea and the influence on the expression of serological inflammatory mediators [ J ] the world journal of integration of Chinese and Western medicine, 2021, 16 (06): 1103-1107; national pharmacopoeia committee the pharmacopoeia of the people' S republic of china 2020 edition (part one) [ S ]. Beijing: china pharmaceutical science and technology Press, 2020].

Modern pharmacological research finds that the Relinqing granules can have a certain treatment effect on gastritis, nephritis, prostatitis and other inflammations, and the inflammation reaction can cause the body to generate heat to different degrees. Traditional Chinese medicine considers that fever is the result of struggle between pathogenic qi and healthy qi after exogenous pathogenic factors invade the body, and is recorded in yellow emperor' S internal classic, which is called fever, and belongs to the category of warm diseases [ Li should exceed, differentiation and treatment of fever in traditional Chinese medicine [ J ] in traditional Chinese medicine journal, 2010, 51 (S1): 125-126; sinoxianxin, yangyiying, senjie, etc. from the point of traditional Chinese medicine, we try to recognize 2019 new type coronavirus pneumonia [ J ]. Tianjin traditional Chinese medicine, 2020, 37 (02): 137-140].

Lipopolysaccharide-induced pneumonia is currently the common pharmacological model for studying pneumonia. Lipopolysaccharide (LPS) is the main component of endotoxin, is derived from the outer membrane of gram-negative bacteria cell wall, and can cause or aggravate a series of clinical symptoms such as asthma, bronchopneumonia and the like after polluted air, occupational dust (cereal powder and the like) and LPS in cigarettes are everywhere and people inhale the substances in certain concentration occupationally and environmentally. After the LPS causes the bronchopneumonia, the bronchopneumonia can be cured through timely and effective treatment, otherwise, the course of disease is prolonged, the repeated attack of airway inflammation can be changed into chronic bronchitis, if the bronchopneumonia is continuously contacted with high-concentration lipopolysaccharide, the condition of the bronchopneumonia is gradually aggravated, and finally, the bronchopneumonia is developed into the pulmonary heart disease. It was found that LPS causes a variety of cells to express chemokines and inflammatory factors at high levels in vitro and in vivo, and that the major cytokines causing increased neutrophil accumulation in lung tissue are IL-1 β and TNF-a.

Therefore, it would be desirable for those skilled in the art to provide a method for determining the efficacy of Relinqing granules in the treatment of pneumonia, and/or to further provide Relinqing granules, and/or to further provide the use of Relinqing granules in the preparation of a medicament for the treatment of pneumonia.

Disclosure of Invention

The invention aims to provide a method for measuring Relinqing granules to treat pneumonia, and/or also provides Relinqing granules, and/or also provides application of the Relinqing granules in preparation of medicines with pneumonia treatment effects. The invention takes Relinqing granules as research objects to research the effect of the Relinqing granules on treating pneumonia of mice with acute pneumonia caused by LPS. It has been surprisingly found that the therapeutic effect of Relinqing granules on pneumonia can be effectively determined by using the method of the present invention. The present invention has been completed based on such findings.

To this end, the present invention provides in a first aspect a method for determining the effect of Relinqing granules on the treatment of pneumonia, or the use of Relinqing granules for the preparation of a medicament for the treatment of pneumonia, or for investigating the effect of Relinqing granules on the treatment of pneumonia by determining the serum TNF- α concentration, said method comprising the steps of:

dividing BALB/c mice into a blank control group, a model group, a Relinqing granule group and a positive control group at random, wherein the male and female halves of the BALB/c mice are respectively;

after the animals are adaptively fed, a LPS atomization method is adopted for molding;

on the next day after molding, the granule group of Relinqing and the positive control group are administered by intragastric administration, and the blank control group and the model group are administered by isopyknic physiological saline;

after administration, blood is taken from eyeballs of mice, the blood is naturally coagulated at room temperature for 30 minutes, centrifuged at 1000Xg for about 15 minutes, serum supernatant is collected and stored at 4 ℃ (or frozen in a refrigerator at-80 ℃);

taking blood from the mice, removing necks of the mice, and killing the mice, and taking alveolar lavage fluid and whole lung tissues to be tested;

determining at least one of: quickly and accurately weighing the weight of the whole lung after the eyeball of the mouse is subjected to blood taking and death, and calculating the lung index;

dehydrating, trimming, embedding, slicing, dyeing, mounting and microscopic examination treatment are carried out on the lung tissue specimen according to a pathological examination standard operation procedure, and pathological changes of the lung tissue are observed;

detection of TNF-alpha, IL-1 beta, LI-6, PGE in serum of mice of each group Using ELISA kit ₂ Content (c);

and obtaining the statistical result of the treatment effect of the Relinqing granules on the (acute) pneumonia mice according to the detection data.

The method according to the first aspect of the present invention, wherein said model is obtained by continuous 3-day molding using LPS nebulization (20 mg/kg) 7 days after adaptive feeding (laboratory environment: 25. + -. 2 ℃ C., relative humidity 50. + -. 5%) of the animal.

The method according to the first aspect of the present invention, wherein the Relinqing granule component is Relinqing granule high dose group (6.24 g/kg), relinqing granule medium dose group (3.12 g/kg), and Relinqing granule low dose group (1.56 g/kg).

The method according to the first aspect of the present invention, wherein dexamethasone acetate is administered orally at 0.878/kg/d in the positive control group.

The method according to the first aspect of the present invention, wherein the high, medium and low dose groups of Relinqing granules and the positive control group are administered by gavage the next day after molding, and the blank control group and the model group are administered by gavage with an equal volume of physiological saline for 1 time/day and are administered continuously for 7 days.

The method according to the first aspect of the present invention, wherein the lung tissue specimen is dehydrated, trimmed, embedded, sliced, stained, mounted, and microscopically processed according to the standard procedures for pathological examination, as follows:

the method according to the first aspect of the present invention, wherein the lung tissue specimen is dehydrated according to a standard procedure for pathological examination under the following conditions: the dehydration time is as follows: 4h for 75% alcohol, 2h for 85% alcohol, 1h for 95% alcohol, 0.5h for 100% alcohol, 0.5h for 100% alcohol, 10min for xylene, 1h for paraffin, 2h for paraffin and 3h for paraffin;

the method according to the first aspect of the present invention, wherein the staining and mounting of the lung tissue specimen according to the standard procedures for pathological examination are carried out as follows: slicing and dewaxing to water, performing hematoxylin staining for 10-20min, washing with tap water for 1-3min, performing hydrochloric acid alcohol differentiation for 5-10s, washing with tap water for 1-3min, placing into warm water or weakly alkaline water solution of 50 ℃ to turn blue until blue appears, washing with tap water for 1-3min, placing into 85% alcohol for 3-5min, performing eosin staining for 3-5min, washing with water for 3-5s, performing gradient alcohol dehydration, performing xylene transparence, and sealing with neutral gum;

the method according to the first aspect of the present invention, wherein the lung tissue specimen is subjected to microscopic examination according to a standard operation procedure for pathological examination, the image acquisition is performed on the sections by using an upright fluorescence microscope, each section is observed for the whole tissue at a magnification of 200, the gross lesion is observed, and the region to be observed is selected to acquire a 400-fold image.

The method according to the first aspect of the present invention, wherein the ELISA detects TNF-. Alpha.IL-1. Beta.LI-6, PGE in the serum of each group of mice ₂ When the content is measured, the serum of each group of rats is taken, and TNF-alpha, IL-1 beta, LI-6 and PGE of the serum of each group of mice are detected according to the operation method of the ELISA kit instruction manual ₂ The content level.

The method according to the first aspect of the present invention, wherein the experimental data are expressed as ± s, the data are analyzed using statistical software (e.g. SPSS), and when the comparisons among the plurality of groups satisfy normal distribution and homogeneity of variance, the analysis is performed by One-way ANOVA,P<0.05 considered the results to be statistically different.

The method according to the first aspect of the present invention, wherein the TNF- α concentration is determined using a TNF- α ELISA kit, comprises the steps of:

a. and (3) diluting the standard: taking 6 small test tubes, numbering in sequence, adding 100 mu l of standard substance diluent into each small test tube, then taking 100 mu l of original concentration standard substance, adding into the first test tube, and fully mixing; then taking 100 microliters of the test tube, adding the test tube into a second test tube, and fully and uniformly mixing; adding 100 microliters of the test tube into a third test tube, and fully and uniformly mixing; then taking 100 microliters of the test tube, adding the test tube into a fourth test tube, and fully and uniformly mixing; adding 100 microliters of the test tube into a fifth test tube, and fully and uniformly mixing; then taking 100 mul from the test tube and discarding; the sixth test tube is used as a No. 0 standard substance, and the concentration of each tube after dilution is respectively as follows: 360pg/ml, 180pg/ml, 90pg/ml, 45pg/ml, 22.5pg/ml, 0pg/ml; standard product holes are formed in the enzyme-labeled coated plate, 50 muL standard products with different concentrations are sequentially added, and each concentration is provided with 5 parallel holes;

b. preparing and adding a sample to be tested: collecting blood with a blood collection tube, naturally coagulating the blood at room temperature for 30 minutes, centrifuging the blood at 1000Xg for about 15 minutes, collecting supernatant (serum), and mixing the supernatant with an additive liquid according to a volume ratio of 9:1, uniformly mixing to obtain a serum sample to be detected; respectively arranging a sample hole to be detected and a blank control hole (the blank control hole is not added with a sample and an enzyme-labeled reagent, and the rest steps are the same as the sample hole to be detected); adding 40 mul of sample diluent into a sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mul of sample to be detected; adding a sample to the bottom of an enzyme label plate hole, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall; the additive liquid is an aqueous solution containing 1.25% of glycine and 0.2% of zinc chloride;

c. and (3) incubation: adding 50 mul of enzyme labeled reagent into each hole, except for a blank control hole; sealing the plate by using a sealing plate film, and then placing the ELISA plate at 37 ℃ for incubation for 30 minutes;

d. washing: diluting 30 times of the concentrated washing liquid by using distilled water to obtain washing liquid; carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;

d. color development: adding 50 mul of color developing agent A into each hole, then adding 50 mul of color developing agent B, lightly shaking and uniformly mixing, and developing for 10 minutes at 37 ℃ in a dark place; then adding 50 μ l of stop solution into each well to stop the reaction, wherein the blue color is changed into yellow color;

e. measurement and calculation: within 15 minutes after adding the stop solution, adjusting the blank control hole to zero, and sequentially measuring the absorbance OD value of each hole by using an enzyme-labeling instrument at the wavelength of 450 nm; and calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, and calculating the concentration of the sample by using the OD value of the sample.

The method according to the first aspect of the present invention, wherein the various materials used are conventional materials.

The method according to the first aspect of the invention, wherein the ELISA kit used is as described in the examples.

Further, the invention relates to a Relinqing granule, which is a granule prepared by taking Polygonum capitatum Buch-ham. Ex D. Don as a raw material.

The heat stranguria clearing granules according to the second aspect of the invention are prepared according to the following method: decocting 1250g of polygonum capitatum in water twice for 1.5 hours each time, filtering the decoction, combining the filtrates, concentrating to a proper amount, filtering, spray-drying, mixing with a proper amount of soluble starch, granulating, and drying to 500 g.

Further, the third aspect of the invention relates to the use of the Relinqing granules described in any one of the second aspects of the invention in the preparation of a medicament for treating pneumonia.

Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are further described below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

Pneumonia refers to inflammation of the terminal airways, alveoli and pulmonary interstitium, and can be caused by pathogenic microorganisms, immune injury, physicochemical factors, allergy and the like. In recent years, scholars at home and abroad often use lipopolysaccharide to induce and establish a pneumonia animal model so as to simulate the pneumonia attack condition. The polygonum capitatum is used for investigating the effect of lipopolysaccharide induced pneumonia animal models on pneumonia treatment.

Polygonum capitatum (Polygonum capitatum Buch-Ham ex D. Don) is a perennial herb of Polygonum of Polygonaceae. Has the efficacies of clearing heat and promoting diuresis, and inducing diuresis for treating strangurtia, and has obvious effect on treating urinary system infection clinically. The Chinese patent medicine preparation 'Relinqing granules' prepared by taking the granules as raw materials enters the national basic medical insurance catalogue in 2004 and enters the basic medicine catalogue in Guizhou province in 2012.

Polygonum capitatum is a common medicine in minority regions and is mainly used for treating pyelonephritis, urinary tract infection, diuresis and stranguria. Related pharmacological studies are rare, and only a few reports exist at present. Experiments are carried out by the optofriend by adopting a mouse bacterial pyelonephritis model, and as a result, WBC and BLD in mouse urine of a polygonum capitatum water extract group are obviously reduced compared with a control group, and the polygonum capitatum water extract has a certain anti-inflammatory effect on pyelonephritis. And (3) performing enterocoelia injection on mice by using the actinomades and the like to observe the death condition of the mice within 5 days, wherein the mortality of a control group is 100 percent, and the mortality of a polygonum capitatum group is 20 percent and 50 percent respectively, and the polygonum capitatum aqueous extract can resist infection caused by escherichia coli. And the wilford et al are administrated to the rabbits by stomach irrigation of the polygonum capitatum aqueous extract, and as a result, compared with a control group, the polygonum capitatum aqueous extract group has no significant difference in body temperature, but can reduce the fever body temperature of the rabbits caused by intravenous injection of typhoid paratyphoid vaccine. And (4) enabling wilford, and the like, respectively performing intragastric administration on the polygonum capitatum aqueous extract to rabbits and mice, and comparing urine volume with a blank group and a fast-urination control group. The result shows that the polygonum capitatum aqueous extract has no obvious diuretic effect on rabbits and mice. Xuyingchun et al used agar dilution to test the in vitro bacteriostatic activity of Polygonum capitatum on 10 strains of Neisseria gonorrhoeae (gonococcus), and as a result, polygonum capitatum has bacteriostatic activity on Neisseria gonorrhoeae. The minimum inhibitory concentration range of the antibacterial agent to 10 strains of gonococcus is 8 to 32g/L, and the average value is 11.2g/L.

The method of the present invention can exhibit one or more excellent effects.

Drawings

FIG. 1: effect of heat stranguria clearing granules on pathological changes of lung tissue in LPS-induced acute pneumonia mice (× 400).

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available.

In the present invention, unless otherwise specified, the heat stranguria clearing granules are prepared according to the following method: decocting 1250g of polygonum capitatum in water twice for 1.5 hours each time, filtering the decoction, mixing the filtrates, concentrating to a proper amount, filtering, spray drying, mixing with a proper amount of soluble starch, granulating, and drying to 500 g. Of course, sugar-containing forms of Relinqing granules may also be used in the present invention. The Relinqing granules meet the regulations of the same-name varieties carried in the 2020 edition Chinese pharmacopoeia.

Example 1: protective effect of Relinqing granules on mice with acute pneumonia caused by LPS (lipopolysaccharide)

The experiment adopts an LPS-induced BALB/c mouse pneumonia model, mice are randomly divided into a normal group, a model group, a positive control group and groups with high, medium and low doses of Relinqing granules, each group comprises 12 mice, the lung index of each group of mice is detected, HE staining is used for detecting the pathological change of the lung tissues of the mice, and ELISA is used for determining the contents of TNF-alpha, IL-1, IL-6 and PGE2 in alveolar lavage fluid.

1. Experimental Material

(1) Laboratory animal

Healthy SPF-grade BALB/c mice, half female, weighing 18-22g, purchased from Schlekschada laboratory animals Co., ltd, hunan, license number: SCXK (Xiang) 2019-0004.

(2) Main instrument

A compressed atomizer (Oubarapi, CNB 69011),

A centrifuge (Nest, 2015002),

Refrigerator (Haier, BCD-215KAN DZ),

A vertical ultra-low temperature storage box (Haier, DW-86L 386),

A pipette (10 to 100 mu l, dalong, KA 0005852),

A pipette (100 to 1000 mu l, dalong, DX 64849),

Beijing Topu (DEM-3, automatic plate washing machine),

BIOBASE (EL 10A, automatic microplate reader).

(3) Primary reagent

Relinqing granules (210602, weimen, guizhou, pharmaceutical Co., ltd.),

Dexamethasone acetate tablet (Changle pharmacy D2104121),

LPS (Lipopolysaccharide, lipopolysaccharide, SIGMA, L9143),

Mouse tumor necrosis factor alpha (TNF-alpha) ELISA Kit (GeneMei, cat number JYM0218 Mo/batch number GR 20220610),

Mouse IL-1 beta ELISA Kit (GeneMei, cat number JYM0531 Mo/batch number GR 20220610),

Mouse IL-6 ELISA Kit (GeneMei, cat number JYM0012 Mo/batch number GR 20220610),

Mouse prostaglandin E2 (PGE 2) ELISA kit (Gene U.S. cat # JYM0603 Mo/lot # GR 20220610).

(4) Reagent configuration

Preparation of LPS solution: 150mg LPS was added to a 15ml volumetric flask and stored in a refrigerator at 4 ℃.

Preparation of a suspension of Relinqing granules: grinding proper amount of Relinqing granules in a mortar, and fixing the volume of 41.6g of ground powder in a volumetric flask of 100ml by using normal saline, wherein the volume is high dosage; taking 40ml of high-dose solution, adding 40ml of normal saline, and shaking up to obtain a medium dose; the medium dose of 20ml solution was taken and 20ml of physiological saline was added and shaken up, this being the low dose.

Preparation of positive control solution: and (3) putting a proper amount of dexamethasone tablets into a mortar for grinding, accurately weighing 8.78g of ground powder, and fixing the volume in a 100ml volumetric flask for standby use at 0.0878 g/ml).

2. Experimental methods

(1) Grouping, modeling and administering drugs

Selecting healthy BALB/c mice, wherein the male and female mice are half of each other and are randomly divided into 6 groups, namely a blank control group, a model group, a Relinqing granule high dose group (6.24 g/kg), a Relinqing granule medium dose group (3.12 g/kg), a Relinqing granule low dose group (1.56 g/kg) and a positive control group (dexamethasone 0.878 mg/kg/d), wherein each group comprises 12 mice;

adaptive feeding (laboratory environment: 25 + -2 deg.C, relative humidity 50 + -5%) for 7 days, and molding; molding by using an LPS atomization method (20 mg/kg), and continuously molding for three days;

on the next day after molding, the groups with high, medium and low doses of Relinqing granules and the positive control group are administered by intragastric administration, and the blank control group and the model group are administered by intragastric administration with physiological saline with the same volume for 1 time/d and are continuously administered for 7d;

(2) Taking materials

The eyeball of the mouse is taken blood and then is removed from the neck to be killed, the blood is naturally coagulated at room temperature for 30 minutes, the blood is centrifuged at 1000Xg for about 15 minutes, serum supernatant is collected and stored at 4 ℃ to be tested (or frozen in a refrigerator at-80 ℃);

after blood is taken from the mouse, alveolar lavage fluid (frozen storage) and whole lung tissues (right leaf frozen storage and left leaf put in formalin for storage) are taken for testing;

(3) Detection and data processing

(3.1) influence of Relinqing granules on lung index of mice with acute pneumonia caused by LPS:

after blood is taken from eyeballs of the mice and the mice die, the weight of the whole lung is quickly and accurately weighed, and the lung index is calculated according to the following formula: lung index = mouse lung weight (g)/mouse body weight (g);

(3.2) influence of Relinqing granules on pathological changes of lung tissues of mice with acute pneumonia caused by LPS:

dehydrating, trimming, embedding, slicing, staining, sealing and microscopic examination the lung tissue specimen according to a standard operation procedure of pathological examination;

[ for example, the operating conditions for dehydrating the fixed lung tissue by the fully automatic dehydrator are: the dehydration time is as follows: 4h for 75% alcohol, 2h for 85% alcohol, 1h for 95% alcohol, 0.5h for 100% alcohol, 0.5h for 100% alcohol, 10min for xylene, 1h for paraffin, 2h for paraffin, 3h for paraffin;

[ for example, the staining and mounting treatment is carried out after embedding and sectioning as follows: slicing and dewaxing to water, staining with hematoxylin for 10-20min, washing with tap water for 1-3min, differentiating with hydrochloric acid and ethanol for 5-10s, washing with tap water for 1-3min, adding into warm water or weakly alkaline water solution at 50 deg.C, returning blue until blue appears, washing with tap water for 1-3min, adding 85% ethanol for 3-5min, staining with eosin for 3-5min, washing with water for 3-5s, dehydrating with gradient ethanol, transparentizing with xylene, and sealing with neutral gum;

for example, in image acquisition, an upright fluorescence microscope (DM 500) manufactured by come card company, germany is used to acquire images of sections, all tissues are observed at 200-fold for each section, gross lesions are observed, 400-fold pictures are acquired of regions to be observed, specific lesions are observed, and lung injury is scored: scoring according to the degree of pathological changes formed by pulmonary interstitial edema, inflammatory cell infiltration, pulmonary edema, pulmonary hemorrhage, pulmonary atelectasis and a transparent membrane according to the normal score, the mild score, the moderate score and the severe score of 0 to 3 respectively ];

(3.3) determination of IL-6, IL-1 beta and PGE in alveolar lavage fluid of mouse with acute pneumonia caused by LPS by Relinqing granules ₂ Effects of TNF- α levels:

detection of TNF-alpha, IL-1 beta, LI-6, PGE in sera of mice of each group by ELISA kit ₂ The content is as follows: taking each group of mouse serum, and detecting IL-1 beta, LI-6 and PGE of each group of serum according to the operation method of the ELISA kit instruction ₂ The content level; the serum was additionally subjected to TNF- α assay according to the method of example 2;

the obtained experimental data are all expressed by + -s, statistical software (SPSS 26.0) is used for analyzing the data, when the comparison among a plurality of groups meets normal distribution and homogeneity of variance, a One-way ANOVA method is adopted for analysis,P<0.05 considered the results to be statistically different;

and obtaining the statistical result of the therapeutic effect of the Relinqing granules on the (acute) pneumonia mice according to the detection data.

3. Results of the experiment

(1) Influence of Relinqing granules on weight and lung index of mice with acute pneumonia caused by LPS (LPS)

The results showed that there was no significant difference in body weight after model building for each group of mice: (P>0.05). The lung index of the model mice is significantly increased compared with that of the blank group (P<0.01 ); the lung index of mice in the high-dose group and the positive control group with Relinqing granules is obviously reduced compared with that in the model group (P<0.05). See table 1.

Table 1: influence of Relinqing granules on weight and pulmonary index of mice with acute pneumonia caused by LPS (plus or minus s, n = 6)

Note: comparison with blank group: ^** P<0.01， ^* P<0.05; comparison with model group: ^## P<0.01， ^# P<0.05；

(2) Influence of Relinqing granules on lung tissue pathology of mice with acute pneumonia caused by LPS (LPS)

HE staining shows that a thin serous membrane is covered on the pleural visceral layer on the surface of the lung tissue of the blank group of mice, a monolayer of mesothelial cells is arranged on the outermost layer, a small amount of fibrous tissues are arranged under the mesothelial cells, the tissue structure is normal, and no obvious thickening exists; branch structures of all levels of bronchus under pleura are complete, and a small amount of inflammatory cell infiltration is occasionally seen in alveolus; the model group can see that a small amount of inflammatory cell infiltration is seen in the bronchus, a large amount of inflammatory cell infiltration is seen in the alveolus, and a small amount of pulmonary hemorrhage is seen; compared with the model group, the pathological changes of the lung tissues of mice in each dose group of the Relinqing granules and the positive control group are obviously reduced, and the lung tissues of the mice in the high dose group of the Relinqing granules are the lightest. The results are shown in FIG. 1. In addition, the pathological results of lung tissues in groups (7) and (8) were found to be similar to those in group (4).

(3) The Relinqing granule can be used for treating mouse pulmonary alveolar lavage fluid IL-6, IL-1 and PGE caused by acute pneumonia caused by LPS ₂ TNF-alpha level

ELISA results showed that model mice had alveolar lavage fluid IL-6, IL-1, PGE, compared to the blank group ₂ Significantly elevated levels of TNF-alpha (A) ((B))P<0.01 ); comparing with model group, the high dose group of Relinqing granule and the positive control group of alveolar lavage fluid IL-6, IL-1, PGE ₂ TNF-alpha level is significantly reduced (P<0.01 Middle and low dose of Relinqing granule, pulmonary alveolar lavage fluid IL-6, IL-1, PGE ₂ Also decreased to different degrees (P<0.05). See table 2.

Table 2: the Relinqing granule can be used for treating mouse pulmonary alveolar lavage fluid IL-6, IL-1 and PGE caused by acute pneumonia caused by LPS ₂ Effects of TNF-alpha levels (± s, n = 6)

Note: comparison with blank group: ^** P<0.01， ^* P<0.05(ii) a Comparison with model groups: ^## P<0.01， ^# P<0.05；

example 2: determination of TNF-alpha concentration in mouse serum

This example 2 uses a TNF- α ELISA kit to detect TNF- α concentrations in mouse serum. The TNF-alpha ELISA kit used in the method is a commercially available JYM0218Mo type mouse tumor necrosis factor alpha (TNF-alpha) ELISA kit, and the TNF-alpha ELISA kit can be used for in-vitro quantitative detection of the content of the tumor necrosis factor alpha (TNF-alpha) in mouse serum, plasma, tissues, cell supernatants and related liquid samples. The specificity of the TNF-alpha ELISA kit is that mouse tumor necrosis factor alpha (TNF-alpha) in a sample can be detected, and no obvious cross reaction exists between the TNF-alpha ELISA kit and other related proteins; the repeatability of the TNF-alpha ELISA kit is that the coefficient of variation in the plate is less than or equal to 9% (usually, the coefficient of variation in the plate is less than or equal to 3% is preferable), the coefficient of variation between plates is less than or equal to 13% (usually, the coefficient of variation between plates is less than or equal to 6% is preferable), the detection range of the kit is 8-500 pg/ml, and the method sensitivity is less than or equal to 1.2pg/ml.

The experimental principle of the TNF-alpha ELISA kit is that a double-antibody sandwich method is used for measuring the level of mouse tumor necrosis factor alpha (TNF-alpha) in a specimen. Coating a microporous plate with a purified mouse tumor necrosis factor alpha (TNF-alpha) antibody to prepare a solid-phase antibody, sequentially adding the TNF-alpha into micropores of the coated monoclonal antibody, then combining with an HRP-labeled TNF-alpha antibody to form an antibody-antigen-enzyme-labeled antibody complex, and adding a substrate TMB for developing color after thorough washing; TMB is converted into blue under the catalysis of HRP enzyme and is converted into final yellow under the action of acid; the shade of the color is positively correlated with the tumor necrosis factor alpha (TNF-alpha) in the sample; the absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the concentration of mouse tumor necrosis factor alpha (TNF-. Alpha.) in the sample was calculated from the standard curve.

The TNF-alpha ELISA kit comprises the following components: the kit comprises 1 part of instruction book, 2 pieces of sealing membrane (96), 1 × 96 pieces of Enzyme label plate (96 wells, 12 wells × 8 strips), 6ml × 1 bottles of Enzyme labeling Reagent (Enzyme Reagent), 0.5ml × 1 bottle of standard (Standards, 720 pg/ml), 1.5ml × 1 bottle of standard diluent, 6ml × 1 bottle of sample diluent, 6ml × 1 bottle of developer A Solution (Substrate Solution A), 6ml × 1 bottle of developer B Solution (Substrate Solution B), 6ml × 1 bottle of Stop Solution (Stop Solution), 6ml × 1 bottle of concentrated washing Solution (Wash Buffer) (20 ml × 30 times) × 1 bottle and 2 pieces of sealing membrane (96).

There are general requirements for treating samples using the TNF-alpha ELISA kits described herein. For serum: collecting blood with blood collecting tube, naturally coagulating at room temperature for 30 min, centrifuging at 1000Xg for about 15 min, collecting serum supernatant, and storing at 4 deg.C or-20 deg.C or-80 deg.C after subpackaging.

This example 2 uses TNF-alpha ELISA kit to detect TNF-alpha concentration in mouse serum, the procedure is as follows:

a. and (3) diluting the standard:

taking 6 small test tubes, numbering in sequence, firstly adding 100 mul of standard substance diluent into each small test tube, then adding 100 mul of original concentration standard substance into the first test tube, and fully and uniformly mixing; then taking 100 mul from the test tube, adding the test tube into a second test tube, and fully and uniformly mixing; then taking 100 mul from the test tube, adding the test tube into a third test tube, and fully and uniformly mixing; then taking 100 microliters of the test tube, adding the test tube into a fourth test tube, and fully and uniformly mixing; adding 100 microliters of the test tube into a fifth test tube, and fully and uniformly mixing; then taking 100 mul from the test tube and discarding; the sixth test tube is used as a No. 0 standard substance, and the concentrations of the test tubes after dilution are respectively as follows: 360pg/ml, 180pg/ml, 90pg/ml, 45pg/ml, 22.5pg/ml, 0pg/ml; standard product holes are formed in the enzyme-labeled coated plate, 50 muL standard products with different concentrations are sequentially added, and each concentration is provided with 5 parallel holes;

b. preparing a sample to be tested and adding sample: blood was collected with a blood collection tube, and the blood was spontaneously coagulated at room temperature for 30 minutes, centrifuged at 1000Xg for about 15 minutes, and the supernatant (serum) was collected and added with an additive in a volume ratio of 9:1, uniformly mixing to obtain a serum sample to be detected; respectively arranging a sample hole to be detected and a blank control hole (the blank control hole is not added with a sample and an enzyme-labeled reagent, and the rest steps are the same as the sample hole to be detected); adding 40 mul of sample diluent into a sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mul of sample to be detected; adding a sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall; the additional liquid is an aqueous solution containing 1.25% of glycine and 0.2% of zinc chloride;

c. and (3) incubation: adding 50 mul of enzyme-labeled reagent into each hole except for a blank control hole; sealing the plate by using a sealing plate film, and then placing the ELISA plate at 37 ℃ for incubation for 30 minutes;

e. measurement and calculation: within 15 minutes after adding the stop solution, adjusting the blank control hole to zero, and sequentially measuring the absorbance OD value of each hole by using an enzyme-labeling instrument at the wavelength of 450 nm; and calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, and calculating the concentration of the sample by using the OD value of the sample. It should be noted that the concentration of TNF- α in the serum sample is calculated by taking into account the dilution factor with the additive.

The results of measuring the serum TNF-alpha concentration of the mice with acute pneumonia induced by LPS of example 1 using the method of this example 2 are shown in Table 2 above (results of 12 animals in each group. + -.s). In addition, in the above table 2, when TNF- α was measured by the method of example 2, 12 animal sera of each group were mixed in equal proportion to obtain a mixed serum (which may be referred to herein as mixed serum X), and the mixed serum was measured by the TNF- α ELISA kit of the method of example 2 to examine the characteristics of the coefficient of variation within a plate (CV within a plate, 5-lane parallel test, hereinafter the same) and the coefficient of variation between plates (CV between plates, 5-lane parallel test, hereinafter the same), and as a result, the repetitive CV within 6 groups of (1) to (6) were: 3.17%, 3.04%, 3.76%, 2.94%, 3.85%, 3.47%, and CV values between 6 groups (1) to (6) are: 5.63%, 6.13%, 6.06%, 5.36%, 5.94%, 6.32%; each group showed excellent reproducibility of the measurement results. However, it has been found that the TNF-alpha ELISA test exhibits significantly worse CV results for sera from animals given hot strangles than the assay requirements of a general ELISA when the above-mentioned additive solution is not premixed with sera, as presented in example 3 and the like below. It should be noted that when other indicators were measured using other ELISA kits in the present invention, such poor results of TNF-. Alpha.CV exhibited only in serum of heat-stranguria-treated animals were not observed.

Example 3: referring to example 1 and example 2, the same TNF- α ELISA kit was used to measure the coefficient of variation between the mouse mixed serum X in the same manner between the plates, except that the additive used in the step "b. Sample preparation and application" of example 2 was an aqueous solution containing 1.25% glycine; as a result, the in-board CVs of 6 groups (1) to (6) were: 3.86%, 3.47%, 3.11%, 16.53%, 14.35%, 14.63%, and CV values between 6 groups (1) to (6) are: 5.66%, 6.13%, 5.85%, 17.34%, 15.31%, 14.13%, showing that sera from mice administered heat strangury were not reproducible for CV characterization in this ELISA method.

Example 4: referring to example 1 and example 2, the same TNF- α ELISA kit was used to detect the intra-and inter-plate coefficient of variation of the mouse mixed serum X in the same method except that the additive used in step "b. Sample preparation and application" of example 2 was an aqueous solution containing 0.2% zinc chloride; as a result, the in-board CV of 6 groups (1) to (6) are: 4.03%, 3.87%, 3.72%, 16.12%, 15.53%, 13.14%, and the CV values between 6 groups (1) to (6) are: 5.32%, 4.82%, 5.11%, 16.78%, 14.63%, 12.11%, showing that the mouse sera given heat strangles had an unsatisfactory reproducibility of CV characterization in this ELISA method.

Example 5: referring to example 1 and example 2, the same TNF- α ELISA kit was used to measure the in-and inter-plate coefficient of variation of the mouse mixed serum X in the same method except that the mixed serum X in the step "b. Preparation of sample to be tested and sample addition" was directly measured without mixing with an additive solution; as a result, the in-board CV of 6 groups (1) to (6) are: 4.63%, 4.13%, 3.93%, 15.89%, 15.23%, 13.74%, and the CV values between 6 groups (1) to (6) are: 5.65%, 5.13%, 5.02%, 17.54%, 16.13%, 13.27%, showing that sera from mice administered heat strangury were not reproducible for CV characterization in this ELISA method.

Example 6: effect of Relinqing granules on treating pneumonia

The invention example 1 investigates and displays the excellent treatment effect of Relinqing granules on related diseases, and the extensive study is carried out on the treatment effect in the example 6.

Example 6 is a test set performed in parallel and added to the test of example 1 (these test sets are not described in example 1, but are additionally described in example 6), i.e., 2 hot-leaching granule test sets of the following numbers (7) and (8) are added:

(7) the dosage group of Relinqing granules is as follows: 3.12g/kg of Relinqing granules and 60mg/kg of ferrous gluconate,

(8) low dose group of Relinqing granules: relinqing granules 1.56g/kg + ferrous gluconate 30mg/kg.

The ferrous gluconate used in the test is a tablet (Kanno pharmaceutical industry) with the national standard H41023259, and during the test, the ferrous gluconate tablet and the Relinqing granules are mixed together in proportion to prepare the drug liquid during the validity period of the drug.

(7) The results of groups (1) and (8) are described in example 1. Also, when TNF- α was measured in example 2, the in-plate CVs of the (7) and (8) groups were 3.44% and 3.09%, respectively, and the inter-plate CVs were 6.42% and 5.84%, respectively, showing excellent methodological characteristics.

The results of this example show that when the Relinqing granules/ferrous gluconate are administered in combination at a ratio of 3.12g/60mg, the ferrous gluconate can enhance the therapeutic effect of the Relinqing granules, particularly the therapeutic effect characterized by the serum concentration of TNF-alpha.

Thus, in any embodiment of any aspect of the invention, the administration of the stranguria reducing granules and the administration of the ferrous gluconate are carried out simultaneously, wherein the weight ratio of the stranguria reducing granules to the ferrous gluconate is 48 to 52:1 for example, the weight ratio of 52:1; for example, in the pharmaceutical use aspect of the present invention, the medicament for treating the corresponding disease further comprises ferrous gluconate in the proportional amount described above.

Polygonum capitatum is the main raw material of Relinqing granules, is the whole herb of Polygonum capitatum of Polygonaceae, is bitter and pungent in taste and mild in nature, and has the effects of clearing heat, removing toxicity and treating heat stranguria through literature records [ Guangxi Chinese herbal medicine, guangxi, edited by health management service station of autonomous region Committee of revolution of Guangxi Zhuang nationality, volume 2 [ M ]. Nanning: guangxi people press 1970]. At present, the research on the Relinqing granules mainly focuses on the treatment of urinary system infection and the like, and a large number of animal experiments and clinical researches prove that the Relinqing granules have obvious curative effect and become one of the first-choice medicines for clinically treating the urinary system infection. Modern researches find that the traditional Chinese medicine composition also has a certain treatment effect on gastritis, nephritis and other inflammations, and the inflammations can cause body fever. The traditional Chinese medicine considers that fever is the result of struggle between pathogenic qi and healthy qi after invasion of exogenous pathogenic factors, and the medicines for clearing the intrinsic heat in the upper jiao are used for relieving the fever of the organism. Most of the traditional Chinese medicines for treating heat stranguria of lower jiao have the effect of clearing heat from upper energizer [ Kangkabusao, willow English, li Shuling, etc.. Professor of Wang Zi uses the method of clearing heat from upper energizer to treat heat stranguria by differentiation [ J ]. Western traditional Chinese medicines 2021, 34 (02): 62-63], so it is presumed that the Relinqing granules have the action of clearing accumulated heat in the upper energizer. In 2020, relinqing granules are brought into the traditional Chinese medicine recovery scheme of New coronary pneumonia in Guizhou province by the traditional Chinese medicine administration in Guizhou province, so that the subject group intends to observe the antipyretic effect of the Relinqing granules and discuss the action mechanism thereof, thereby providing more choices for clinic.

Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria, consisting of lipid a, core polysaccharide and an O-specific chain. The core oligosaccharide is connected with an O-specific chain with a hydrophilic structure at the outer layer and a lipoid A with hydrophobicity at the inner layer. Wherein the core oligosaccharide is a branched oligosaccharide chain consisting of 9 to 10 glycosyl groups, and can be further divided into inner core oligosaccharide and outer core oligosaccharide. The inner core oligosaccharide attaches the core oligosaccharide to lipid a via an acid-labile ketosidic bond. The outer core oligosaccharide, which is composed mainly of neutral and basic hexoses, is linked to an O-specific chain, and is the basis for determining the core type of LPS, with differences in monosaccharide composition and configuration among different strains. When bacteria invade the human body, they release their surface LPS, which first binds to lipopolysaccharide-binding protein (LBP), which transports LPS to the membrane surface of immune cells, binding to the membrane surface protein CD 14. LPS is not released in the normal living state of bacteria, but is released after the death and rupture of bacterial thalli, artificial lysis or active growth and reproduction of cells, has NO toxic effect, is used as a non-specific immunogen, and is interacted with host effector cells (mainly monocytes, macrophages and neutrophils) after entering microcirculation to secrete bioactive molecules such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (lL-6), reactive Oxygen Species (ROS), NO and the like, so that clinical syndromes such as body fever, disseminated intravascular coagulation, multi-organ failure, shock and the like are caused. The toxicity of LPS is to form a protective barrier around bacteria by LPS to avoid the action of antibiotics, and act on host cells to generate inflammatory cytokines, so that the internal environment of the organism is in a disordered state, and diseases such as endotoxemia and sepsis are caused. The LPS has immune activation, and has various biological functions of resisting tumor, resisting radiation, resisting infection, promoting generation of after cataract, promoting periodontal inflammation, promoting proliferation of pericyte, relieving asthma, etc.

In summary, the invention discusses the protection effect and mechanism of Relinqing granules on mice with acute pneumonia caused by LPS from the experimental point of view. The specific method of the invention is that LPS is adopted to induce BALB/c mouse pneumonia model, mice are randomly divided into blank group, model group, positive control group and groups with high, medium and low doses of Relinqing granules, each group is 12, lung index of each group of mice is detected, HE staining is used for detecting pathological change of lung tissues of the mice, and ELISA is used for detecting contents of TNF-alpha, IL-1, IL-6 and PGE2 in alveolar lavage fluid. In the experiment, the lung index of the model group mice and IL-6, IL-1 and PGE in the alveolar lavage fluid ₂ The TNF-alpha content is obviously increased compared with that of a blank group, and the lung index and the IL-6, IL-1 and PGE in the alveolar lavage fluid of the mice are treated by high dose of the Relinqing granules ₂ The content of TNF-alpha is obviously reduced compared with that of a model group; mouse alveolar lavage fluids IL-6, IL-1, PGE after administration of Medium and Low dose therapy with Relinqing granules ₂ The reduction of different degrees also indicates that the Relinqing granules can inhibit the generation of inflammatory factors, thereby playing the role of treating pneumonia, and the treatment effect has dose-effect relationship in a certain range.

As can be seen from the results herein, the stranguria-treating granule reduces the symptoms of pneumonia such as acute pneumonia and improves the related serum indicators, and the results indicate that the use of stranguria-treating granule is effective in treating pneumonia.

The embodiments are only for illustrating the composition and efficacy of the invention, and not for limiting the scope of the invention, therefore, it will be apparent to those skilled in the art that similar modifications can be made without departing from the structure of the invention, and all such modifications are within the scope of the invention. These should also be construed as the scope of the present invention, and they should not be construed as affecting the effectiveness of the practice of the present invention or the applicability of the patent.

Claims

1. A method for determining the effect of Relinqing granules on treating pneumonia or a method for investigating the effect of Relinqing granules on treating pneumonia by determining serum TNF-alpha concentration, wherein the method comprises the following steps:

after administration, blood is taken from the eyeballs of the mice, the blood is naturally coagulated at room temperature for 30 minutes, centrifuged at 1000Xg for about 15 minutes, serum supernatant is collected and stored at 4 ℃ to be tested (or frozen in a refrigerator at-80 ℃);

taking blood from the mouse, removing neck, killing, and taking alveolar lavage fluid and whole lung tissue to be tested;

dehydrating, trimming, embedding, slicing, dyeing, mounting and microscopic examination are carried out on the lung tissue specimen according to a pathological examination standard operation procedure, and the pathological change of the lung tissue is observed;

detection of TNF-alpha, IL-1 beta, LI-6, PGE in serum of mice of each group Using ELISA kit ₂ The content;

2. The method according to claim 1, wherein said model is obtained by continuous 3-day modeling using LPS nebulization (20 mg/kg) 7 days after the animal is acclimatized.

3. The method of claim 1, wherein:

the granule components of Relinqing are Relinqing granule high dose group (6.24 g/kg), relinqing granule medium dose group (3.12 g/kg), and Relinqing granule low dose group (1.56 g/kg);

dexamethasone acetate is orally taken to be 0.878/kg/d in the positive control group;

on the next day after molding, the groups with high, medium and low doses of Relinqing granules and the positive control group were administered by intragastric administration, and the blank control group and the model group were administered by intragastric administration with physiological saline of equal volume for 1 time/d and continuously administered for 7d.

4. The method of claim 1, wherein:

the operation conditions for dehydrating the lung tissue specimen according to the standard operation procedure of pathological examination are as follows: the dehydration time is as follows: 4h for 75% alcohol, 2h for 85% alcohol, 1h for 95% alcohol, 0.5h for 100% alcohol, 0.5h for 100% alcohol, 10min for xylene, 1h for paraffin, 2h for paraffin, 3h for paraffin;

the lung tissue specimen is stained and mounted according to the standard operation procedure of pathological examination according to the following operations: slicing and dewaxing to water, staining with hematoxylin for 10-20min, washing with tap water for 1-3min, differentiating with hydrochloric acid and ethanol for 5-10s, washing with tap water for 1-3min, adding into warm water or weakly alkaline water solution at 50 deg.C, returning blue until blue appears, washing with tap water for 1-3min, adding 85% ethanol for 3-5min, staining with eosin for 3-5min, washing with water for 3-5s, dehydrating with gradient ethanol, transparentizing with xylene, and sealing with neutral gum;

when the lung tissue specimen is microscopically inspected according to a pathological examination standard operation program, an upright fluorescence microscope is adopted to collect images of the sections, each section firstly observes all tissues under 200 times, general lesions are observed, and a region to be observed is selected to collect 400 times of images.

5. The method of claim 1, wherein: ELISA detection of TNF-alpha, IL-1 beta, LI-6, PGE in serum of each group of mice ₂ When the content is measured, the serum of each group of rats is taken, and TNF-alpha, IL-1 beta, LI-6 and PGE of the serum of each group of mice are detected according to the operation method of the ELISA kit instruction manual ₂ The content level.

6. The method of claim 1, wherein the experimental data are expressed in ± s, the data are analyzed using statistical software (e.g., SPSS), and when the comparisons among the plurality of groups satisfy normal distribution and homogeneity of variance, the analysis is performed by One-way ANOVA,P<0.05 considered the results to be statistically different.

7. The method according to claim 1, wherein the TNF- α concentration is determined using a TNF- α ELISA kit comprising the steps of:

a. and (3) diluting the standard: taking 6 small test tubes, numbering in sequence, adding 100 mu l of standard substance diluent into each small test tube, then taking 100 mu l of original concentration standard substance, adding into the first test tube, and fully mixing; then taking 100 mul from the test tube, adding the test tube into a second test tube, and fully and uniformly mixing; then taking 100 mul from the test tube, adding the test tube into a third test tube, and fully and uniformly mixing; then taking 100 mul from the test tube, adding the test tube into a fourth test tube, and fully and uniformly mixing; then taking 100 mul from the test tube, adding the test tube into a fifth test tube, and fully and uniformly mixing; then taking 100 mul from the test tube and discarding; the sixth test tube is used as a No. 0 standard substance, and the concentration of each tube after dilution is respectively as follows: 360pg/ml, 180pg/ml, 90pg/ml, 45pg/ml, 22.5pg/ml, 0pg/ml; standard product holes are formed in the enzyme-labeled coated plate, 50 muL standard products with different concentrations are sequentially added, and each concentration is provided with 5 parallel holes;

b. preparing a sample to be tested and adding sample: collecting blood with a blood collection tube, naturally coagulating the blood at room temperature for 30 minutes, centrifuging the blood at 1000Xg for about 15 minutes, collecting supernatant (serum), and mixing the supernatant with an additive liquid according to a volume ratio of 9:1, uniformly mixing to obtain a serum sample to be detected; respectively arranging a sample hole to be detected and a blank control hole (the blank control hole is not added with a sample and an enzyme-labeled reagent, and the rest steps are the same as the sample hole to be detected); adding 40 mul of sample diluent into a sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mul of sample to be detected; adding a sample to the bottom of an enzyme label plate hole, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall; the additional liquid is an aqueous solution containing 1.25% of glycine and 0.2% of zinc chloride;

c. incubation: adding 50 mul of enzyme labeled reagent into each hole, except for a blank control hole; sealing the plate by using a sealing plate film, and then placing the enzyme label plate at 37 ℃ for incubation for 30 minutes;

e. measurement and calculation: within 15 minutes after adding the stop solution, adjusting to zero by using a blank reference hole, and sequentially measuring the absorbance OD value of each hole by using an enzyme-labeling instrument under the wavelength of 450 nm; and calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, and calculating the concentration of the sample by using the OD value of the sample.

8. A granule for treating stranguria, RELINQING granule, is prepared from herba Polygoni Capitati (Polygonum capitatum Buch. -ham. Ex D. Don).

9. Relinqing granules according to claim 8, which are prepared according to the following process: decocting 1250g of polygonum capitatum in water twice for 1.5 hours each time, filtering the decoction, combining the filtrates, concentrating to a proper amount, filtering, spray-drying, mixing with a proper amount of soluble starch, granulating, and drying to 500 g.

10. Use according to claim 8 or 9 in the manufacture of a medicament for the treatment of pneumonia.